Thyroid auto‐antibodies in newly diagnosed multiple sclerosis patients: A cross sectional study

Abstract Introduction Multiple sclerosis (MS) is a chronic inflammatory disease in which the immune system attacks the myelin sheath of the central nervous system (CNS). It has been proposed that autoimmune conditions may occur together and an individual's immune system may attack more than one system. Autoimmune thyroid disease is one of the most common comorbidities along with MS. Since thyroid hormones are crucial for normal brain function and remyelination, we aimed to determine the prevalence of thyroid dysfunction in a group of MS patients compared with healthy controls. Methods This cross‐sectional study was conducted in medical clinics affiliated to Shiraz University of Medical Sciences, South of Iran. To prevent the effects of MS modifying drugs on thyroid function, we examined 73 newly diagnosed MS patients, which had not been treated yet, compared to 72 healthy individuals. Results After measurement of the serum level of TSH, Anti TPO‐Ab, and Anti TG‐Ab, we found a significantly higher prevalence rate of abnormal TSH levels (high or low) in the MS group (p = 0.02). We also found a higher frequency of thyroid dysfunction in the female MS group (p = 0.01). However, there was no significant difference in the two other anti‐thyroid antibodies among the groups. Our results demonstrate a significant and positive linear relationship between age and TSH levels (R = 0.402; p < 0.001) and also age and Anti TPO‐Ab levels (R = 0.397; p < 0.001) among the MS population. Conclusion We found a higher prevalence of TSH alteration among the MS population. Anti TPO‐Ab and Anti TG‐Ab levels did not differ among groups. These findings suggest that MS patients might be at an increased risk for thyroid dysfunction. However, further studies are required to determine the underlying cause. The linear relationship between age and TSH and Anti TPO‐Ab levels in MS patients suggest that there is an association between TSH dysfunction and age.


| BACKGROUND
MS is an inflammatory disease of the CNS that damages both oligodendrocytes and axons leading to diverse symptoms. 1The exact cause of this disease is not yet determined, however numerous findings point to the roles of both genetic predisposition and environmental factors and the disease has been generally accepted as an autoimmune condition. 2udies have shown that people with one autoimmune disease are susceptible to developing another one and that autoimmune diseases may occur together. 3About 25% of MS patients are diagnosed with at least one other autoimmune disorder.Hashimoto thyroiditis, psoriasis, irritable bowel disease, and rheumatoid arthritis are some of the comorbidities with MS. 4,5 A notable correlation exists between autoimmune thyroid diseases (ATD) and MS, affirming the heightened risk of thyroid disorders in individuals with MS. 6 Thyroid-mediated signaling is essential in the process of myelination and remyelination and thyroid hormone administration has been shown to improve myelin repair. 7e to the important role of thyroid hormones in the normal process of myelin repair, many studies have investigated the prevalence of thyroid dysfunction in MS patients.
Other than that, fatigue, muscle weakness, and myalgia are common symptoms in both MS and hypothyroidism, 8 therefore a coexisting ATD, which is the most common cause of hypothyroidism could be missed.That being said, due to the common symptoms in both MS and ATD and the role of thyroid hormones in remyelination, it is crucial to evaluate thyroid function when MS patients present with such symptoms.
To better understand any correlation between MS and ATD we conducted this study in Shiraz, south of Iran, to compare the prevalence of autoimmune thyroid auto-antibodies in MS patients with the control group.To prevent the effects of MS-modifying drugs on thyroid function, we selected our patients among those who had not yet received disease-modifying treatments for MS.We examined serum levels of Thyroid Stimulating Hormone (TSH), Anti Thyroid Peroxidase Antibody (Anti TPO-Ab) and Anti-Thyroglobulin Antibody (Anti TG-Ab) in MS patients and the control group.

| Study design and patients
The present study is a cross-sectional study that was conducted in a descriptive and analytical manner.All experiments were accepted by the Ethics Committee of Shiraz University of Medical Sciences (SUMS).In this study, we investigated the prevalence of autoimmune thyroid diseases in patients with MS.The research participants included all newly diagnosed MS patients based on McDonald's 2017 MS diagnosis criteria and who had not received any treatment for MS except methylprednisolone 9 and were referred to all medical centers affiliated to SUMS, Shiraz, Iran, within a period of 6 months.The control group was the patients who had referred to these centers due to headaches during this period and who had no history of any other disease.Written informed consent was obtained from all individuals enrolled in this study.

| Data collection
Patient information was collected using a checklist.The checklist consists of two parts.The first part is related to demographic characteristics, including age, sex, marital status, etc.The second part is related to the patient's clinical and paraclinical information, including the serum level of Anti TPO-Ab and Anti TG-Ab and TSH.

| Blood samples
We referred the MS patients and the control group to the laboratory.
After an overnight fasting, venous blood samples were taken and centrifuged at 3,000 rpm for 10 min, and samples were stored at −80°C until analysis.The concentration of anti-thyroid antibodies (Anti-TPO or Anti-TG) and TSH were measured with the immunochemiluminometric assay (ICMA).The reference ranges were 0.3-5.3µIU/mL for TSH, 0-35 IU/mL for Anti-TPO, and 0-115 IU/mL for Anti-TG.One-step Immunoradiometric assay (IRMA) was used for measuring TSH as recommended by manufacturers (Siemens).Among the participants, 96 people (66.2%) were women and 49 people (33.8%) were men.The frequency of females in the MS patient group was 74% and, in the control group was 58.3%.The difference in gender between the control group and the patients was statistically significant (p = 0.04).

| Statistical analysis
Among MS patients, 17 individuals (23.3%) displayed total abnormal TSH levels-either higher or lower than the normal range.In contrast, 7 individuals (9.7%) in the control group showed abnormal TSH levels.
Although we found that a higher percentage of MS patients had abnormal Anti-TG (8.2%) and abnormal Anti-TPO (16.4%) compared to the control group, difference among groups did not meet standard statistical significance (see Table 1).
Table 2 shows subgroup analysis of TSH, Anti TG-Ab, and Anti TPO-Ab in female patients with MS compared with the female control group.According to the results, the frequency of abnormal TSH levels in female patients with MS was more than that of females in the control group, and this observed difference was statistically significant (p = 0.01).The proportion of abnormal Anti TG-Ab in females from the control group exceeded that of female patients with MS.
Conversely, the percentage of abnormal Anti TPO-Ab in female MS patients surpassed that of females in the control group, although no significant differences were observed (p = 0.96, p = 0.51 respectively).Table 3 demonstrates comparison of TSH, Anti TG-Ab, and Anti TPO-Ab in the male MS patients group compared to the male control group.According to the results, the percentage of abnormal TSH in male patients with MS was more than that of men in the control group (26.3% vs 16.6%), but the differences were not statistically significant (p = 0.41).The percentage of both abnormal Anti TG-Ab, and Anti TPO-Ab of males with MS was higher than the control group but the differences observed were not statistically significant (p = 0.73, p = 0.54 respectively).
Pearson's correlation analysis between age and TSH levels/Anti TPO-Ab in the MS patients and control groups showed that there were significant linear and positive relationship between the age of the patients and their TSH (R = 0.402; p < 0.001) and Anti TPO-Ab (R = 0.397; p < 0.001) levels (Figures 1 and 2).However, this relationship was weak and not significant in the control groups (R = 0.091; p = 0.44), (R = 0.095; p = 0.42) respectively.
The correlation analysis between age and Anti TG-Ab levels in the MS patients showed a nonsignificant, weak linear and inverse correlation (R = -0.004;p = 0.48).Moreover, no significant correlation was found for the control group (R = 0.037; p = 0.37).

| DISCUSSION
Among the many comorbidities that may be observed with MS, ATD is one of the most common autoimmune conditions reported in MS patients. 11However, in some instance, thyroid dysfunction seems to T A B L E 1 Serum level of TSH, Anti TG-Ab and Anti TPO-Ab in the MS patients and control groups.| 3 of 6 be a result of disease modifying therapies (DMT) such as alemtuzumab and interferon-beta. 12,13It is important to evaluate whether thyroid dysfunction in MS patients is independent from DMT and to establish its true cause among these individuals. 14The most common finding in MS patients is a positive Anti TPO-Ab with a normal functioning thyroid. 15Previous studies have examined thyroid function among the MS population.Results of these studies vary, some studies found no thyroid dysfunction among MS and control groups while some studies demonstrated an increased rate of thyroid dysfunction in MS patients which will be discussed further. 6In the present study, we measured TSH levels, along with Anti-TG Ab and Anti-TPO Ab.According to the findings of previous studies, the prevalence of thyroid dysfunction in MS patients ranges between 6.44% and 11%. 11,16In our study, we found that 23.3% of MS patients had abnormal TSH levels which was significantly higher than the control group (9.8%).Our results demonstrated that among genders, TSH abnormality is higher in both male and female MS group compared to the same gender of the control group.Although this difference in men was not statistically significant.Autoimmunity is considered the most frequent etiology of thyroid dysfunction.A very important part of the genetic contribution to autoimmunity is gender. 17 Data were analysed using SPSS version 26.0 (IBM, New York, NY, USA).All results were presented as median and interquartile range (IQR) for quantitative variables and frequency (%) for qualitative variables.The Kolmogorov-Smirnov test was used to check for normality of distribution.The differences in categorial variables including gender, abnormal or normal levels of TSH, Anti-TG, and Anti-TPO were assessed using the two-tailed Pearson's Chi-square test.The two-tailed Mann-Whitney U test was used for age difference.Pearson's correlation coefficient was used to analyze the relationship between age and serum TSH/Anti TPO-Ab/Anti TG-Ab levels.p < 0.05 was used to indicate a significant difference.The results were interpreted and reported following the guidelines set forth by Assel et al. 10 3 | RESULTS A total of 72 patients with MS and 73 healthy controls have been evaluated.The median age of subjects was 30, IQR:25-35.The median age of the MS patients was 30, IQR: 25-35, and the median age of the control group was 31, IQR: 27-35.We found no significant difference in age among the participants (p = 0.28).
L Durelli et al. demonstrated that high or low TSH levels did F I G U R E 1 Correlation between age and TSH concentration in the MS group.There is a linear and positive correlation between the age of the patients and their TSH concentration (R = 0.402, p < 0.001).F I G U R E 2 Correlation between age and Anti TPO-Ab concentration in the MS group.There is a linear and positive correlation between age and serum Anti TPO-Ab in the MS group (R = 0.397, p < 0.001).
Serum level of TSH, Anti TG-Ab and Anti TPO-Ab in the female MS patients and control groups.Serum level of TSH, Anti TG-Ab, and Anti TPO-Ab in the male MS patients and control groups.